Method of assay of target nucleic acid

ABSTRACT

A simple and accurate method for assay of a single-stranded RNA containing a specific nucleic acids sequence in a sample at almost constant temperature by using at least the following reagents (A) to (I), which comprises a step of adding the reagents (A) to (I) one by one (in any order), in combinations of at least two or all at once and  
     a step of measuring a fluorescent signal in the presence of the reagent (I) at least once after addition of at least the reagents (A) to (H);  
     (A) a first single-stranded oligonucleic acid complementary to a sequence neighboring the 5′ end of the specific nucleic acids sequence in the single-stranded RNA,  
     (B) a second single-stranded oligo DNA complementary to a 3′-end sequence within the specific nucleic acids sequence,  
     (C) an RNA-dependent DNA polymerase,  
     (D) deoxyribonucleoside triphosphates,  
     (E) a third single-stranded oligo DNA having (1) a promoter sequence for a DNA-dependent RNA polymerase, (2) an enhancer sequence for the promoter and (3) a 5′-end sequence within the specific nucleic acids sequence, in this order from the 5′ end,  
     (F) a DNA-dependent DNA polymerase,  
     (G) a DNA-dependent RNA polymerase,  
     (H) ribonucleoside triphosphates, and  
     (I) a fourth single-stranded oligo DNA complementary to the specific nucleic acids sequence which is labeled so that it gives off a measurable fluorescent signal on hybridization with a nucleic acid containing the specific nucleic acids sequence.

[0001] The present invention relates to a method for assay of asingle-stranded RNA containing a specific nucleic acids sequence in asample, which enables detection and quantification of viral RNA andbacterial mRNA and is effective in diagnosis of infectious diseases andin judging the effects of therapeutic agents for infectious diseases.The present invention also related to a method for producing largeamounts of DNA and RNA containing a specific nucleic acids sequence,which is useful for cloning useful genes and exploring unknown genes.The present invention further relates to a reagent set for use in thesemethods.

[0002] Assays of biogenic components require high specificity andsensitivity. Sequence-specific hybridizability of a nucleic acid with acomplementary nucleic acid (a nucleic acid probe) is utilized in assaysof a specific nucleic acid.

[0003] Measurable signaling corresponding to the amount to thehybridization product is essential for quantification of a targetnucleic acids sequence. In clinical diagnosis, high-sensitive signalingis required because samples usually contain traces of target nucleicacids.

[0004] For quantification of a target nucleic acid in a sample, a methodwhich involves solid phase hybridization of the target nucleic acid witha labeled nucleic acid probe which gives off measurable signals onmembranes, beads or gels, called sandwich assay, has been employed. Inpractice, two nucleic acid probes specific for different sequenceswithin the target nucleic acid are used: a first nucleic acid probelabeled with a dye of a visible color, a fluorescent substance or anenzyme which catalyzes production of such a dye or a fluorescentsubstance, and a second probe immobilized on a solid phase. These probesare added to a sample and hybridize with the specific nucleic acid inthe sample, forming a complex on the solid phase. Then, the reactionmixture is separated into the supernatant and the solid phase to removethe unhybridized first probe (B/F separation). The specific nucleic acidin the sample can be determined by measurement of the label on the solidphase. When the label is an enzyme which catalyzes production of a dyeof a visible color or a fluorescent substance, a precursor of the dye orfluorescent substance is added as the substrate of the enzyme to thesample solution after the B/F separation, and the resulting dye orfluorescent substance is measured.

[0005] Especially, in diagnosis of virus infections, because clinicalsamples usually contains the target nucleic acid (viral nucleic acid) intrace amounts, sandwich assay using a chemiluminescent substance as thesubstrate of the enzyme or preceded by preamplification of the targetnucleic acid in samples by polymerase chain reaction (PCR) has beenattempted to increase the signal intensity and sensitivity for thepurpose of sensitive and reproducible assay.

[0006] Another PCR-based method known for assay of a target nucleic acidis competitive PCR. In the method, PCR is performed in the presence of agiven concentration of a nucleic acid having a base sequence similar tothat of the target nucleic acid (a competitor) in the sample, theconcentration of the target nucleic acid is estimated from theamplification level of the target nucleic acid. In practice, a sample issubjected to PCR in the presence of various concentrations of a nucleicacid which has a terminal sequence complementary to a primer and isdistinguishable from the amplification product from the target nucleicacid (for example, by the length) by some separation means such aselectrophoresis simultaneously.

[0007] Homogeneous support-free assay for PCR-based quantification of atarget nucleic acid has also been proposed. For example, the presentinventors reported an assay method wherein the initial amount of thetarget nucleic acid is determined by fluorescence measurement of thereaction solution after each PCR cycle during PCR in the presence of afluorescent intercalative dye (JP-A-5237000; Igaku-no-Ayumi, 173(12),959-963 (1995); Analytical Biochemistry, 229, 207-213 (1995)). In theassay method, as the PCR amplification products are double-stranded DNA,a fluorescent intercalative dye which changes its fluorescencecharacteristic for example, by increasing the fluorescence intensity,upon intercalation into double-stranded nucleic acid, is added to samplesolutions prior to PCR amplification, and the fluorescence intensity ofthe reaction solution is monitored to determine the initial amount ofthe target nucleic acid from the pattern of the fluorescenceenhancement. Further, this method makes it possible to keep track ofamplification of the target nucleic acid by fluorometric measurement ofthe reaction solution in a closed reaction vessel and therefore canobviate the problem of false positive results attributable to carryoverof the amplification products because sampling of the reaction solutionfrom the reaction vessel is unnecessary.

[0008] At present, the detection or quantification limit of the sandwichassay of a target nucleic acid is at most about 10⁵ copies even if thefist probe labeled with multiple enzyme molecules is used to produce alarge amount of a luminescent substance in an enzymaticchemiluminescence system known for a relatively high sensitivity,because the first probe non-specifically adsorbed on the solid supportgives off a considerable background signal (background) and thereforeproduces errors in the measurement of the solid phase hybridization onthe surface.

[0009] In order to prevent non-specific adsorption of a first probe on asolid support, hydrophilic surface treatment of the support, blocking ofthe adsorptive sites on the support surface with protein, throughwashing of the solid support after the B/F separation and use of a highdetergency cleaning solution containing a surfactant have beenattempted.

[0010] However, chemical hydrophilic surface treatment is not applicableto some kinds of supports depending to the material of the support andcan be technically difficult. Also, protein coating of the supportsurface for blocking of the adsorption sites on the support can lead toa different type of non-specific adsorption due to interaction betweenthe protein coat and the nucleic acid segment or the label of the firstprobe. Washing operations after the B/F separation can not be increasedindefinitely due to operational limitations, and the surfactant added toa cleaning solution can induce decomposition of the hybrid formed on thesupport.

[0011] In competitive PCR assay, it is necessary to perform PCR onsample solutions containing a competitor at various concentrationsranging over the predicted target nucleic acid concentration foranalysis of one sample. Besides, post-PCR separation of sample solutionswithdrawn from the reaction vessels, for example, by electrophoresis isnecessary. Therefore, competitive PCR assay is difficult to automate andinappropriate for clinical diagnosis which requires speedy handling of agreat number of samples. Further, due to the need to withdraw samplesolutions from reaction vessels, the false positive problem attributableto carryover of the amplification products in practical application ofthe PCR assay remais to be solved.

[0012] PCR-based assays in the presence of a fluorescent intercalativedye have a problem that when the sample contains a large amount of otherdouble-stranded DNA such as genomic DNA in addition to the specificnucleic acid, the intercalative dye intercalates into otherdouble-stranded DNA producing a significant background, because it isbased on the ability of a fluorescent intercalative dye to intercalateinto double-stranded nucleic acid. Further, in PCR-based assays using apair of oligo DNAs complementary to the specific nucleic acid sequenceas the primers for chain elongation, the primers can hybridize eachother, depending on their base sequences, and elongate by using eachother as templates to produce a primer dimer. Because the intercalationof a fluorescent intercalative dye into double-stranded nucleic acid isnot specific, production of such a primer dimer creates a problem of ahigh background.

[0013] The demand for automation in clinical diagnostics likelycontinues to increase to realize speedy and reproducible analyses of agreat number of samples. PCR involves repetitious rapid heating andcooling of reaction solutions and entails strict temperature controlduring heating and cooling because accuracy and reproducibility of theseoperations can affect the results of PCR. However, it is not easy toprovide a full-automatic instrument having enough incubation ability tosatisfy these requirements and a sufficient throughput capacity.

[0014] Further, in the case of RNA as viral nucleic acids in mostviruses, PCR is preceded by synthesis of cDNA by reverse transcriptaseusing RNA as the template, and therefore virtually two steps areinvolved.

[0015] For amplification of a target nucleotide at constant temperature,a so-called NASBA method is known. The NASBA method seems easy toautomate because heating or cooling is unnecessary, but requiressandwich assay or electrophoretic separation of the amplified RNA andtherefore can not solve the problems attributable to these operations.

[0016] Accordingly, the object of the present invention is to provide asimple and accurate method for assay of a single-stranded RNA containinga specific nucleic acids sequence in a sample at almost constanttemperature without repetitious rapid heating and cooling of reactionsolutions in PCR or using a support in measurement of the amplified RNA,wherein all the operations are preferably done in a closed vessel.Another object of the present invention is to provide a simple methodfor producing a nucleic acid having a specific nucleic acids sequence atalmost constant temperature.

[0017] The present inventors developed a nucleic acid probe which iscomplementary to a specific nucleic acids sequence in the target nucleicacid and labeled with a fluorescent intercalative dye so as to give ameasurable fluorescent signal on binding to the target nucleic acid(JP-A-7-185599/EP-A-714986/Nucleic Acid Research, 24(24), 4992-4997(1996)). The nucleic acid probe gives a measurable fluorescent signal onhybridization with the target nucleic acid and therefore enablesdetection of hybridization and quantification of the hybridizationproduct without separation of the unhybridized probe. Further, thepresent inventors have established synthesis of an RNA having thespecific acid sequence by using a nucleic acid polymerase and nucleicacid primers in the presence of the nucleic acid probe at constanttemperature, namely amplification and assay of the target RNA atconstant temperature without using a support preferably in a closedsystem, and have accomplished the present invention.

[0018] According to claim 1 of the present application, the presentinvention provides a simple and accurate method for assay of asingle-stranded RNA containing a specific nucleic acids sequence in asample at almost constant temperature by using at least the followingreagents (A) to (I), which comprises a step of adding the reagents (A)to (I) one by one (in any order), in combinations of at least two or allat once and

[0019] a step of measuring a fluorescent signal in the presence of thereagent (I) at least once after addition of at least the reagents (A) to(H);

[0020] (A) a first single-stranded oligonucleic acid complementary to asequence neighboring the 5′ end of the specific nucleic acids sequencein the single-stranded RNA,

[0021] (B) a second single-stranded oligo DNA complementary to a 3′-endsequence within the specific nucleic acids sequence,

[0022] (C) an RNA-dependent DNA polymerase,

[0023] (D) deoxyribonucleoside triphosphates,

[0024] (E) a third single-stranded oligo DNA having (1) a promotersequence for a DNA-dependent RNA polymerase, (2) an enhancer sequencefor the promoter and (3) a 5′-end sequence within the specific nucleicacids sequence, in this order from the 5′ end,

[0025] (F) a DNA-dependent DNA polymerase,

[0026] (G) a DNA-dependent RNA polymerase,

[0027] (H) ribonucleoside triphosphates, and

[0028] (I) a fourth single-stranded oligo DNA complementary to thespecific nucleic acids sequence which is labeled so that it gives off ameasurable fluorescent signal on hybridization with a nucleic acidcontaining the specific nucleic acids sequence.

[0029] According to claim 21 in the present application, the presentinvention provides a simple method for producing a nucleic acid having aspecific nucleic acids sequence at almost constant temperature by usingat least the following reagents (A) to (H), which comprises a step ofadding the reagents (A) to (G) one by one (in any order), incombinations of at least two or all at once to a single-stranded DNAhaving (1) a promoter sequence for a DNA-dependent RNA polymerase, (2)an enhancer sequence for the promoter and (3) the specific nucleic acidssequence, in this order from the 5′ end or to a double-stranded DNAconsisting of the single-stranded DNA and a complementary DNA strand anda step of measuring a fluorescent signal from the reagent (H) at leastonce after addition of at least the reagents (A) to (G);

[0030] (A) a single-stranded oligo DNA complementary to a 3′-endsequence within the specific nucleic acids sequence,

[0031] (B) an RNA-dependent DNA polymerase,

[0032] (C) a DNA-dependent DNA polymerase,

[0033] (D) deoxyribonucleoside triphosphates,

[0034] (E) a DNA-dependent RNA polymerase,

[0035] (F) ribonucleoside triphosphates,

[0036] (G) a single-stranded DNA having (1) a promoter sequence for aDNA-dependent RNA polymerase, (2) an enhancer sequence for the promoterand (3) a 5′-end sequence within the specific nucleic acids sequence, inthis order from the 5′ end,

[0037] (H) a fourth single-stranded labeled oligo DNA complementary tothe specific nucleic acids sequence which gives a measurable fluorescentsignal on hybridization with a nucleic acid containing the specificnucleic acids sequence.

[0038] According to claims 24 to 28 in the present application, thepresent invention also provides a reagent or reagent set for performingthe above-mentioned method, and specifically according to claim 24, thepresent invention provides a reagent set for performing the methodaccording to claim 1 or 21, which comprises at least

[0039] a first reagent containing the first single-stranded oligonucleicacid,

[0040] a second reagent containing tris-acetate, magnesium acetate,potassium acetate, sorbitol and dimethyl sulfoxide,

[0041] a third reagent containing dithiothreitol, deoxyribonucleosidetriphosphates, ribonucleoside triphosphates, bovine serum albumin, thesecond single-stranded oligo DNA and the third single-stranded oligoDNA,

[0042] a fourth reagent containing an RNA-dependent DNA polymerase, aDNA-dependent DNA polymerase, a DNA-dependent RNA polymerase and anRNase inhibitor and a fifth reagent containing the fourthsingle-stranded oligo DNA.

[0043] Specifically according to claim 25, the present inventionprovides a reagent set for performing the method according to claim 1 or21, which comprises at least

[0044] a first reagent containing the first single-stranded oligonucleicacid,

[0045] a second reagent containing tris-acetate, magnesium acetate,potassium acetate, sorbitol and dimethyl sulfoxide,

[0046] a third reagent containing dithiothreitol, deoxyribonucleosidetriphosphates, ribonucleoside triphosphates, bovine serum albumin, thesecond single-stranded oligo DNA, the third single-stranded oligo DNAand the fourth single-stranded oligo DNA and

[0047] a fourth reagent containing an RNA-dependent DNA polymerase, aDNA-dependent DNA polymerase, a DNA-dependent RNA polymerase and anRNase inhibitor.

[0048] According to claim 26, the present invention further provides areagent set for performing the method according to claim 1 or 21, whichcomprises at least

[0049] a first reagent containing the first single-stranded oligonucleicacid,

[0050] a second reagent containing tris-acetate, magnesium acetate,potassium acetate, sorbitol and dimethyl sulfoxide,

[0051] a third reagent containing dithiothreitol, deoxyribonucleosidetriphosphates, ribonucleoside triphosphates, bovine serum albumin, thesecond single-stranded oligo DNA and the third single-stranded oligoDNA,

[0052] a fourth reagent containing the fourth single-stranded oligo DNA,an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, aDNA-dependent RNA polymerase and an RNase inhibitor.

[0053] Specifically according to claim 27, the present inventionprovides a reagent for performing the method according to claim 1 or 21,which comprises at least the first single-stranded oligonucleic acid,the second single-stranded oligo DNA, the third single-stranded oligoDNA, the fourth single-stranded oligo DNA, an RNA-dependent DNApolymerase, a DNA-dependent DNA polymerase, a DNA-dependent RNApolymerase, deoxyribonucleoside triphosphates, ribonucleosidetriphosphates, tris-acetate, magnesium acetate, potassium acetate,sorbitol, dimethyl sulfoxide, dithiothreitol, bovine serum albumin andan RNase inhibitor, namely a single reagent obtained by mixing them all.

[0054]FIG. 1 shows the results of the 2% agarose gel electrophoresis ofthe product of the PCR using the second and third single-stranded oligoDNAs in Example 1. Lanes 2 to 4: known concentrations of the standardDNA, lanes 5 to 7: 0.2 to 5 μl of the PCR product.

[0055]FIG. 2 shows the results of the 2% agarose gel electrophoresisafter the reactions at various magnesium acetate concentrations inExample 2. The arrow indicates the specific product (about 300 bp). Theconcentrations of magnesium acetate are expressed in terms of finalconcentration.

[0056]FIG. 3 shows the results of the 2% agarose gel electrophoresisafter the reactions at various potassium acetate concentrations inExample 3. The arrow indicates the specific product (about 300 bp). Theconcentrations of potassium acetate are expressed in terms of finalconcentration.

[0057]FIG. 4 shows the results of the 2% agarose gel electrophoresisafter the reactions at various sorbitol concentrations in Example 4. Thearrow indicates the specific product (about 300 bp).

[0058]FIG. 5 shows the results of the 2% agarose gel electrophoresisafter the reactions in the presence of various concentrations of thestandard DNA in Example 5. The arrow indicates the specific product(about 300 bp).

[0059]FIG. 6 shows the results of the electrophoresis on a polyacrylamide gel containing 12% urea after the reaction of the 133mer RNA, thefirst single-stranded oligo DNA complementary to a neighboring sequenceat the 5′ end of the specific nucleic acids sequence within the 133merRNA and various concentrations of RnaseH in Example 6. The arrowsindicate the 133mer and the 72mer.

[0060]FIG. 7 shows the results of the 2% agarose gel electrophoresisafter the reactions in the presence of various concentrations of thestandard RNA in Example 7. The arrow indicates the specific product(about 300 bp).

[0061]FIG. 8 shows the results of the 2% agarose electrophoresis of theproducts of the reactions using various concentrations of the standardRNA (10⁶ copies/5 μl) after RNaseA or DNaseI treatment in Example 8. TheDNA product and the RNA product are pointed.

[0062]FIG. 9 shows the results of densitometric quantification of theproducts from the standard RNA (10⁶ copies/5 μl) at various reactiontimes after RNaseA or DNaseI treatment followed by 2% agarose gelelectrophoresis in Example 9.

[0063]FIG. 10 shows the results of the 2% agarose gel electrophoresis ofthe products from the standard RNA (10⁶ copies/5 μl) at various reactiontimes in Example 10.

[0064]FIG. 11 shows the results of fluorescence measurement afteraddition of the fourth single-stranded oligo DNA to the products shownin FIG. 10 obtained in Example 10.

[0065]FIG. 12 shows the results of the fluorescence measurement aftervarious times of reactions using the standard RNA (10⁶ copies/5 μl) inthe presence of the fourth single-stranded oligo DNA in Example 11.

[0066]FIG. 13 shows the results of the fluorescence measurement aftervarious times of reactions using the standard RNA (10⁶ copies/5 μl) inthe presence of the fourth single-stranded oligo DNA having a modified3′ end (having ddTTP at the 3′ end) in Example 11.

[0067]FIG. 14 shows the results of the 2% agarose gel electrophoresisafter after various times of reactions using the standard RNA (10⁶copies/5 μl) in the presence of the fourth single-stranded oligo DNAhaving a modified 3′ end (having ddTTP at the 3′ end) in Example 12.

[0068]FIG. 15 shows the results of the densitometric quantitativeanalysis of the electrophoretogram shown in FIG. 14 in Example 12.

[0069]FIG. 16 shows the results of the fluorescence measurement aftervarious times of reactions using the standard RNA (10⁶ copies/5 μl) inthe presence of the fourth single-stranded oligo DNA having a modified3′ end (having ddTTP at the 3′ end) in Example 13.

[0070]FIG. 17 shows the results of the fluorescence measurement aftervarious times of reactions using the standard RNA (10⁴, 10⁵ and 10⁶copies/5 μl) in the presence of the fourth single-stranded oligo DNAhaving a modified 3′ end (having ddTTP at the 3′ end) in Example 14.

[0071]FIG. 18 shows the plot of the fluorescence enhancement at areaction time of 3 hours against the initial concentration of thestandard RNA based on the amplification profiles shown FIG. 17 obtainedin Example 14.

[0072]FIG. 19 shows the structure of the fourth single-stranded oligoDNA used in Examples, YO-271. The DNA moiety on the left and thefluorescent intercalative dye, oxazole yellow on the right are linkedvia the linker shown in this figure so that the fluorescent dye canintercalates into a double strand upon formation of the double strand bythe DNA moiety.

[0073] Now, the present invention will be described in detail.

[0074] According to claim 1 of the present application, the presentinvention provides a method for assay of a single-stranded RNAcontaining a specific nucleic acids sequence in a sample (a target RNA).Herein, assay means both qualitative assay of the RNA in a sample andquantitative assay of the RNA in a sample.

[0075] The specific nucleic acids sequence means a base sequence withinthe single-stranded RNA which starts at the 5′ end with the sequence (3)in the after-mentioned third single-stranded oligo DNA and end at the 3′end with a sequence complementary to the after-mentioned secondsingle-stranded oligo DNA. The specific nucleic acids sequence can bedefined arbitrarily but must contain a sequence specific enough todistinguish the target RNA from other nucleic acids. In particular, whena sequence sufficiently distinguishable from nucleic acids other thanthe target nucleic acid by its 5′ and 3′ ends is selected as thespecific nucleic acids sequence, the specificity of the synthesis of asingle-stranded RNA having the specific nucleic acids sequence accordingto the present invention improves.

[0076] Further, when the specific nucleic acids sequence furthercontains an internal sequence which sufficiently distinguishes thetarget nucleic acid from other nucleic acids, the specificity of theassay according to the present invention further improves.

[0077] In the present invention according to claim 1, when the targetRNA is present in the sample, a large amount of an RNA having thespecific nucleic acids sequence is synthesized eventually.

[0078] The reagent (A) is a first oligonucleic acid complementary to aneighboring sequence at the 5′ end of the specific nucleic acidssequence within the target RNA. The reagent allows the target RNA to becut at the 5′ end of the specific nucleic acids sequence so that onbinding of a second single-stranded oligo DNA to the target RNA, anRNA-dependent DNA polymerase synthesizes cDNA from the resulting RNAfragment having the specific nucleic acids sequence at the 5′ end as thetemplate in the presence of the after-mentioned reagents (B) to (D) togive a cDNA having a 3′-end sequence complementary to the specificnucleic acids sequence. For example, when a DNA as the firstoligonucleic acid is added together with RNase H, the target RNA is cutwhere the first oligonucleic acid binds to the target RNA, so as to havethe first base of the specific nucleic acids sequence at the 5′ end.Otherwise, a DNAzyme or ribozyme which catalytically cuts asingle-stranded nucleic acid at a specific site (Proc. Natl. Acad. Sci.USA, 94, 4262-4266(1997)) may be used as the first oligonucleic acid.

[0079] RNase H non-specifically cleaves an RNA in an RNA-DNAhetero-duplex. Therefore, when RNase H is used, the RNase H is virtuallydeactivated, for example, by heating or by addition of a known RNase Hinhibitor prior to addition of the reagent (B). In the case of heating,it is satisfactory to raise the temperature to 60-70° C. and keep thattemperature for quite a short time. Accordingly, when RNase H is used,after the reagent (A) and RNase H are added separately orsimultaneously, an appropriate time of incubation precedes addition ofthe reagents (B) to (I). However, a DNAzyme or a ribozyme as the reagent(A) can be added alone or at the same time as other reagents without theneed of heating or addition of an inhibitor.

[0080] The reagent (B) is a second single-stranded oligo DNAcomplementary to a 3′-end sequence within the specific nucleic acidssequence. The reagent (B) hybridizes with the target RNA so that anRNA-dependent DNA polymerase synthesizes cDNA from the RNA as thetemplate in the presence of the after-mentioned reagents (C) to (D) togive a cDNA having a 5′-end sequence complementary to a 3′-end sequencewithin the specific nucleic acids sequence. The second oligonucleic acidis added in an amount of 0.02 to 1 μM in the presence of the targetnucleic acid.

[0081] The reagent (C) is an RNA-dependent DNA polymerase, and thereagent (D) is the substrate of the reagent (C), deoxyribonucleosidetriphosphates. In the presence of the reagents (B) to (D), a cDNA havinga 5′-end sequence complementary to the 3′ end of the specific nucleicacids sequence within the target RNA is synthesized.

[0082] To the cDNA in the form of a DNA-RNA double strand with thetemplate RNA, the ability to hybridize with a third single-strandedoligo DNA as the after-mentioned reagent (E) is imparted. For thispurpose, for example, 5 to 20% of dimethyl sulfoxide (hereinafterreferred to as DMSO) is added to make the complementary binding betweenthe DNA and RNA less tight. DMSO does not interfere with the actions ofthe other reagents and acts satisfactory only if it is added so as tocoexist with the DNA-RNA double strand at least prior the DNA synthesisby the reagents (D) to (F). Alternatively, because some RNA-dependentDNA polymerases represented by avian myoblastoma virus polymerase(hereinafter AMV reverse transcriptase) cut RNA in a DNA-RNA doublestrand, though with low activities, such an enzyme with an RNA degradingaction can be used as the above-mentioned reagent (C). It isparticularly preferable to use the action of DNSO and the action of anRNA-dependent DNA polymerase such as CMV reverse transcriptase, althoughit is satisfactory to use the action of either one. By the actions ofDMSO and/or an RNA-dependent DNA polymerase, the RNA in the RNA-DNAdouble strand is degraded and/or separated, leaving a single-strandedDNA.

[0083] The reagent (E) is a third single-stranded oligo DNA having (1) apromoter sequence for a DNA-dependent RNA polymerase, (2) an enhancersequence for the promoter and (3) a 5′-end sequence within the specificnucleic acids sequence in this order from the 5′ end. The third oligoDNA is added in an amount of 0.02 to 1 μM in the presence of the targetnucleic acid. The region (3) in the third oligo DNA binds to the 3′ endof the DNA synthesized in the presence of the reagents (B) to (D).Therefore, in the presence of the reagents (D) and (F), a DNA-dependentDNA polymerase complementarily elongates the 3′ end of the third DNAusing the DNA as the template and the 3′ end of the DNA by using thethird oligo DNA as the template to give a complete double-stranded DNA.

[0084] The reagent (F) is a DNA-dependent DNA polymerase. In the presentinvention, it is particularly preferred to use fewer kinds of reagentsby using a single enzyme which acts both as the RNA-dependent DNApolymerase as the reagent (C) and as the DNA-dependent DNA polymerase.For example, AMV reverse transcriptase, which has the activities of thetwo polymerases, is particularly preferable for use in the presentinvention because it also degrades the RNA chain in a DNA-RNA doublestrand as described above and is also commercially available.

[0085] The reagent (G) is a DNA-dependent RNA polymerase, and thereagent (H) is the substrate of the reagent (G), ribonucleosidetriphosphates. The double-stranded DNA synthesized in the presence ofthe reagents (D) to (F) has a promoter region for the DNA-dependent RNApolymerase at one end. Therefore, in the presence of the reagents (G)and (H), the synthesis of the DNA is immediately followed by synthesisof a single-stranded RNA having the specific nucleic acids sequence.Specific examples of the DNA-dependent RNA polymerase as the reagent (G)include T7 RNA polymerase, T3 polymerase and SP6 RNA polymerase.

[0086] The single-stranded RNA synthesized in the presence of thereagents (G) to (H) has the specific nucleic acids sequence. Therefore,coexistence of the synthesized single-stranded RNA with the reagents (B)to (F) sets off the above-mentioned series of reactions again from thestart. Thus, in the present invention, it is possible to synthesizeabove-mentioned double-stranded DNA having a promoter region at one endfrom a trace of the target RNA in the sample only by adding therespective reagents to the sample without any hardly automatableoperations such as heating, and the double-stranded DNA gives rise tosynthesis of a single-stranded RNA having the specific nucleic acidssequence. The synthesized single-stranded RNA participates in the nextround of synthesis of the double-stranded DNA. Consequently, thesingle-stranded RNA having the specific nucleic acids sequencedrastically increases with the elapse of time.

[0087] The rate of synthesis of the single-stranded RNA having thespecific nucleic acids sequence and the final amount of the synthesizedsingle-stranded RNA depend on the amount of the target RNA in thesample. Accordingly, the use of the reagent (I) in the present inventionenables determination of the target RNA in the sample.

[0088] The reagent (I) is a fourth single-stranded labeled oligo DNAcontaining the sequence complementary to the specific nucleic acidssequence which gives a measurable fluorescent signal on hybridizationwith a nucleic acid containing the specific nucleic acids sequence. Thefourth oligo DNA may be, for example, a fluorescent intercalativedye-linked DNA. The DNA moiety is from 6 to 100 nucleotides long,preferably from 10 to 30 nucleotides long, to secure the specificity forthe specific nucleic acids sequence in the assay. Of course, the DNAmoiety must be complementary to a sequence within the specific nucleicacids sequence which sufficiently distinguishes the target nucleic acidfrom other nucleic acids.

[0089] The DNA moiety preferably has a 3′-end sequence which isuncomplementary to the specific nucleic acids sequence or has achemically modified 3′ end so that the 3′ end does not elongate by theaction of the already existing RNA-dependent DNA polymerase onhybridization with the synthesized single-stranded RNA having thespecific nucleic acids sequence.

[0090] If the DNA moiety is hybridized with another nucleic acid, thefluorescent intercalative dye intercalates into the resulting doublestrand and changes its fluorescence characteristic. For this purpose,the fluorescent intercalative dye may be linked to the DNA moiety via alinker of an appropriate length. Any linker that does not hinder thefluorescent intercalative dye from intercalating into the double strandmay be used without any particular restriction. A bifunctionalhydrocarbon linker having functional groups at both ends is particularlypreferable for the easiness of its use in modification ofoligonucleotides. Alternatively, a commercial kit (C6-Thiolmodifier,tradename, Clonntech) may be used.

[0091] The fluorescent intercalative dye is not particularly limited aslong as it changes the fluorescent characteristic, for example emitsfluorescence having a different peak wavelength, on intercalation into adouble strand. However, those which enhance the fluorescence onintercalation are particularly preferable in view of the easiness offluorescence measurement. More specifically, particularly preferablefluorescent intercalative dyes are, for example, thiazole orange,oxazole yellow and their derivatives, because they show radical changein the fluorescence.

[0092] The fluorescent intercalative dye may be linked to any sites ofthe DNA moiety, including the 5′ end, the 3′ end and the middle, as longas the linkage neither hinders the fluorescent intercalative dye fromintercalating into a double strand nor hinders the DNA moiety fromhybridizing with RNA.

[0093] The reagent (I) gives off a measurable fluorescent signal in thepresence of the single-stranded RNA having the specific nucleic acidssequence synthesized in the presence of the double-stranded DNA, thereagents (G) and (H). Surprisingly, the synthesized single-stranded RNAhas been found to serve as a template for DNA synthesis in the presenceof the reagents (B) to (D) even when the hybrid of the RNA and thefourth oligo DNA is giving off a fluorescent signal. In other words, inthe present invention, a series of events, synthesis of DNA from RNA,synthesis of double-stranded DNA and synthesis of RNA fromdouble-stranded DNA in the presence of the respective reagents, takeplace in the presence of the fourth oligo DNA.

[0094] Therefore, the target nucleic acid in a sample can be determinedby measuring the fluorescent signal from the reagent (I) which is addedafter addition of the reagents (A) to (H) at least once. The reagent (I)may be added at the same time as the other reagents because the presenceof the fourth oligonucleic acid is not an obstacle to synthesis of asingle-stranded RNA having the specific nucleic acids sequence afterall.

[0095] When the fluorescent signal is measured only once, prior to themeasurement, addition of the reagents (A) to (H) is followed bysufficient time of incubation for synthesis of the single-stranded RNAhaving the specific nucleic acids sequence. The reagent (I) must beadded before the measurement, for example, at the same time as the otherreagents. This style of measurement is called an end point assay, andthe target nucleic acid in a sample can be determined, for example, fromcomparison with the results obtained by performing the same procedure onsolutions containing the known amounts of the target nucleic acid. Inthe present invention, the addition of the reagents (A) to (H) ispreferably followed by durational measurement of fluorescent signalsimmediately or after a certain time lag. Though during synthesis of thesingle-stranded RNA, the fourth DNA binds to and separates from thesynthesized RNA repeatedly, but it is possible to monitor the increaseof the single-stranded RNA because the measured fluorescent signals fromthe bound fourth DNA correlate with the amounts of the RNA at themoments of the measurements. The measurement may be done continuously orintermittently at constant intervals. Thus, the time course of thefluorescent signal can be traced by durational measurement, and theamount (initial amount) of the target RNA in a sample can be determined,for example, from the time required to obtain a stable fluorescentsignal after addition of the reagents (A) to (H) or the time lag betweenthe addition of the reagents (A) to (H) and drastic increase of thefluorescent signal.

[0096] As will be demonstrated later in Examples, in the presentinvention, all the above-mentioned reagents (A) to (I) are preferred tobe free from chlorides. Further, it is preferred to use an acetate suchas magnesium acetate or potassium acetate in addition to the reagents(A) to (H). An acetate is added at latest at the time of synthesis ofsingle-stranded DNA in the presence of the reagents (B) to (D)preferably at a concentration of from 5 to 20 mM for magnesium acetate,from 50 to 200 mM for potassium acetate. In the present invention, it ispreferred to further use sorbitol. Sorbitol is added at latest at thetime of synthesis of single-stranded DNA in the presence of the reagents(B) to (D). Further, use of protein such as bovine serum albumin and areducing agent such as dithiothreitol, use of an RNase inhibitor with aview to inhibiting degradation of the synthesized single-stranded RNA byRNase are also preferred. Still further, a buffering agent is alsopreferably used so as to keep the reaction solution within an active pHrange for the respective enzymes to be used. As the buffering agent,tris-acetate is particularly preferable. These reagents are added atlatest at the time of synthesis of single-stranded DNA in the presenceof the reagents (B) to (D). Use of these optional reagents leads toincrease of the amount of the single-stranded RNA synthesized as theproduct.

[0097] The method of the present invention enables assay of a target RNAin a sample at constant temperature without heating. The constanttemperature is may be any temperature at which the respectiveoligonucleic acids used as the reagents (A), (B), (E) and (I) in thepresent invention can hybridize, and the enzymes as the reagents (C),(F) and (F) are active, without any restriction. Specifically speaking,the temperature is selected from the range of from 35 to 60° C. Thetemperature may not be exactly constant, and it is sufficient to keepthe temperature almost constant.

[0098] As is evident from the above explanation, in the assay methodaccording to claim 1, the reagents to be used may be added one by one,in combination of at least two or all at once. In particular, when allthe reagents are added to a sample all at once, assay of the target RNAin a closed vessel can be realized to solve the problem of carryover ofthe synthesized single-stranded RNA which can happen when the vessel isopened or closed.

[0099] According to claim 21 of the present application, the presentinvention provides a simple method for producing a nucleic acid having aspecific nucleic acids sequence at almost constant temperature by usingat least the following reagents (A) to (G), which comprises a step ofadding the reagents (A) to (G) one by one (in any order), incombinations of at least two or all at once to a single-stranded DNAhaving (1) a promoter sequence for a DNA-dependent RNA polymerase, (2)an enhancer sequence for the promoter and (3) the specific nucleic acidssequence in this order from the 5′ end or to a double-stranded DNAconsisting of the single-stranded DNA and a complementary DNA and a stepof measuring a fluorescent signal from the reagent (H) at least once inthe presence of at least the reagents (A) to (G);

[0100] (A) a single-stranded oligo DNA complementary to a 3′-endsequence in the specific nucleic acids sequence,

[0101] (B) an RNA-dependent DNA polymerase,

[0102] (C) a DNA-dependent DNA polymerase,

[0103] (D) a deoxyribonucleoside triphosphate,

[0104] (E) a DNA-dependent RNA polymerase,

[0105] (F) a ribonucleoside triphosphate,

[0106] (G) a single-stranded DNA having (1) a promoter sequence for aDNA-dependent RNA polymerase, (2) an enhancer sequence for the promoterand (3) a 5′-end sequence in the specific nucleic acids sequence in thisorder from the 5′ end,

[0107] (H) a fourth single-stranded labeled oligo DNA containing thesequence complementary to the specific nucleic acids sequence whichgives a measurable fluorescent signal on hybridization with a nucleicacid containing the specific nucleic acids sequence.

[0108] As is understandable from the explanation already made, in themethod according to claim 23, a nucleic acid having a specific nucleicacids sequence is produced by using the double-stranded DNA from whichthe single-stranded RNA is synthesized in the method according to claim1, as the starting material. The specific nucleic acids sequence usedherein does not have to be attributed to the target RNA, unlike thespecific nucleic acids sequence in claim 1.

[0109] The double-stranded DNA as the starting material can be preparedby known methods using PCR or a DNA synthesizer. The first oligo DNA asthe reagent (A) hybridizes with the single-stranded DNA having (1) apromoter sequence for a DNA-dependent RNA polymerase, (2) an enhancersequence for the promoter and (3) the specific nucleic acids sequenceand elongates using the DNA as the template in the presence of thereagents (A), (C) and (D) to give the double-stranded DNA, which servesas the starting material. The double-stranded DNA as the startingmaterial can also be prepared by performing the respective operationsmentioned for the method according to claim 1 on a single-stranded RNAcontaining the specific nucleic acids sequence. The single-stranded DNAas the reagent (A), the single-stranded oligo DNA as the reagent (G) andthe single-stranded oligo DNA as the reagent (H) correspond to thesecond oligo DNA, the third oligo DNA and the fourth oligo DNA in themethod according to claim 1. Therefore, the modes of addition andactions of these reagents, preferable examples of the respectivepolymerases, optional reagents other than (A) to (H) and the procedurecan be easily understandable by referring to the previous explanation.In short, a single-stranded RNA having the specific nucleic acidssequence is synthesized from the double-stranded DNA as the startingmaterial in the presence of the reagents (E) and (F), then from thesingle-stranded RNA a single-stranded DNA complementary to the specificnucleic acids sequence is synthesized in the presence of the reagents(E) and (F), and from the single-stranded DNA the double-stranded DNA asthe starting material is synthesized in the presence of the reagents (C)and (D). In this method, the reagent (A) used in the method according toclaim 1 and the operations associated with the reagent (A) such asheating are unnecessary in this method unless the double-stranded DNA asthe starting material is prepared from a single-stranded RNA whichcontaining the specific nucleic acids sequence somewhere other than the5′ end.

[0110] When the fluorescent signal is measured only at once, themeasurement is done in the presence of the reagent (I) after sufficienttime of incubation following the addition of the reagents (A) to (G).When the fluorescent signal is measured over a period of time, thedurational measurement follows addition of the reagents (A) to (G)immediately or after a certain time lag. Once production of apredetermined amount of a nucleic acid having the specific nucleic acidssequence is indicated by the measured fluorescent signal or the timecourse of the fluorescent signal, then the produced nucleic acid isextracted.

[0111] To obtain a single-stranded RNA having the specific nucleic acidssequence as the final product, RNA extraction is preceded by addition ofan enzyme which degrades DNA such as DNase. To obtain a single-strandedDNA containing the specific nucleic acids sequence or a double-strandedDNA consisting of such a DNA single strand and a complementary strand asthe final product, DNA extraction is preceded by addition of an enzymewhich degrades RNA such as RNase. Of course, a mixture of such an RNAand such a DNA can be obtained by extraction without addition of anuclease. DNA and/or RNA can be easily extracted by known techniquessuch as ethanol precipitation.

[0112] The present invention according to claim 1 or 21 described abovecan be performed, for example, by using a reagent set which comprises atleast a first reagent containing a first single-stranded oligonucleicacid, a second reagent containing tris-acetate, magnesium acetate,potassium acetate, sorbitol and dimethyl sulfoxide, a third reagentcontaining dithiothreitol, deoxyribonucleoside triphosphates,ribonucleoside triphosphates, bovine serum albumin, a secondsingle-stranded oligo DNA and a third single-stranded oligo DNA, afourth reagent containing an RNA-dependent DNA polymerase, aDNA-dependent DNA polymerase, a DNA-dependent RNA polymerase and anRNase inhibitor and a fifth reagent containing a fourth single-strandedoligo DNA, and by adding these reagents in numerical order to a sample.When the present invention according to claim 21 does not require thefirst single-stranded oligonucleic acid, the first reagent can beomitted.

[0113] The present invention according to claim 1 or 21 described abovecan also be performed, for example, by using a reagent set whichcomprises at least a first reagent containing a first single-strandedoligonucleic acid, a second reagent containing tris-acetate, magnesiumacetate, potassium acetate, sorbitol and dimethyl sulfoxide, a thirdreagent containing dithiothreitol, deoxyribonucleoside triphosphates,ribonucleoside triphosphates, bovine serum albumin, a secondsingle-stranded oligo DNA, a third single-stranded oligo DNA and afourth single-stranded oligo DNA and a fourth reagent containing anRNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, aDNA-dependent RNA polymerase and an RNase inhibitor, and by adding thesereagents in numerical order to a sample. When the present inventionaccording to claim 21 does not require the first single-strandedoligonucleic acid, the first reagent can be omitted.

[0114] The present invention according to claim 1 or 21 described abovecan also be performed, for example, by using a reagent set whichcomprises at least a first reagent containing a first single-strandedoligonucleic acid, a second reagent containing tris-acetate, magnesiumacetate, potassium acetate, sorbitol and dimethyl sulfoxide, a thirdreagent containing dithiothreitol, deoxyribonucleoside triphosphates,ribonucleoside triphosphates, bovine serum albumin, a secondsingle-stranded oligo DNA and a third single-stranded oligo DNA, afourth reagent containing a fourth single-stranded oligo DNA, anRNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, aDNA-dependent RNA polymerase and an RNase inhibitor, and by adding thesereagents in numerical order to a sample. When the present inventionaccording to claim 21 does not require the first single-strandedoligonucleic acid, the first reagent can be omitted.

[0115] The present invention according to claim 1 or 21 described abovecan also be performed, for example, by using a reagent which comprisesat least a first single-stranded oligonucleic acid, a secondsingle-stranded oligo DNA, a third single-stranded oligo DNA, a fourthsingle-stranded oligo DNA, an RNA-dependent DNA polymerase, aDNA-dependent DNA polymerase, a DNA-dependent RNA polymerase,deoxyribonucleoside triphosphates, ribonucleoside triphosphates,tris-acetate, magnesium acetate, potassium acetate, sorbitol, dimethylsulfoxide, dithiothreitol, bovine serum albumin and an RNase inhibitor,and by adding the reagent to a sample. When the present inventionaccording to claim 21 does not require the first single-strandedoligonucleic acid, the first single-stranded oligo DNA can be omitted.The reagent is a single reagent containing all the requiredconstituents, unlike the other reagent sets. Therefore, it workssatisfactory when added to a sample only once.

[0116] The concentration of each constituent in the above-mentionedreagent sets or reagent should be adjusted so that each constituent ispresent in an required amount in a sample when added to the sample. Asdescribed previously, a single enzyme having the actions of bothRNA-dependent DNA polymerase and DNA-dependent DNA polymerase may beused as the RNA-dependent DNA polymerase and DNA-dependent DNApolymerase.

[0117] Now, the present invention will be described in further detail byreferring to Examples. However, the present invention should be by nomeans is restricted to these specific Examples.

EXAMPLE 1

[0118] Preparation of Double-stranded DNA

[0119] A double-stranded DNA consisting of a DNA single strand havingthe SP6 promoter sequence at the 5′ end and a complementary DNA singlestrand, was prepared

[0120] 70 μl of a reaction solution having the following composition waspored into a PCR tube.

[0121] 10.7 mM tris-HCl buffer (pH 8.3)

[0122] 53.6 mM potassium chloride

[0123] 2.36 mM magnesium chloride

[0124] 0.268 mM each of dATP, dGTP, dCTP and dTTP

[0125] 0.257 μM third single-stranded oligo DNA =p1 (sequence) 5′ ATTTAG GTG ACA CTA TAG AAT ACA ACA CTC CAC CAT AGA TCA CTC CCC TG 3′

[0126] 0.257 μM second single-stranded oligo DNA

[0127] (sequence) 5′ ACT CGC AAG CAC CCT ATC A 3′

[0128] 0.032 U/μl commercially available DNA-dependent DNA polymerase(Ampli Taq, tradename, Perkin Elmer)

[0129] Then, 5 μl of a 1,865-bp double-stranded DNA (10⁻⁶ copies)containing bases 1 to 1,863 in human hepatitis C virus (HCV) cDNA wasadded as a standard DNA. For the base numbers, literature (Kato et al,.Proc. Natl. Sci., USA, 1990, 87, 9524-9528) should be referred to.

[0130] Then, the reaction solution was heated and incubated at 95° C.for 9 minutes, and an incubation cycle of the following steps (1) to (3)were repeated 40 times.

[0131] (1) Incubation at 95° C. for 30 seconds,

[0132] (2) Incubation at 65° C. for 30 seconds and

[0133] (3) Incubation at 72° C. for 1 minute.

[0134] After 40 cycles of incubation, the reaction solution waswithdrawn and subjected to 2% agarose electrophoresis followed byethidium bromide staining.

[0135]FIG. 1 shows the results on the gel stained with ethidium bromide.FIG. 1 clearly shows single bands of DNA of about 320 bp., indicatingthat preparation of a DNA having the following base sequence containingthe SP6 promoter sequence (underlined) at the 5′ end and a complementarystrand by the above-mentioned procedure.

[0136] (sequence) 5′ ATT TAG GTG ACA CTA TAG AAT ACA A-(bases 11 to 300in HCV cDNA) 3′

EXAMPLE 2

[0137] Effects of Magnesium Acetate Concentration

[0138] The optimum concentration of magnesium acetate for the presentinvention was studied. Reaction solutions having the followingcomposition were prepared, and 14 μl of the reaction solutions werepored into PCR tubes.

[0139] 85.7 mM tris-acetate (pH 8.1)

[0140] 16.1, 32.1 or 48.2 mM magnesium acetate

[0141] 214.3 mM potassium acetate

[0142] 21.4% DMSO

[0143] 32.1% sorbitol

[0144] 2.1 mM each of ATP, GTP, CTP and UTP

[0145] 2.1 mM each of dATP, dGTP, dCTP and dTTP

[0146] 214 μg/ml BSA

[0147] 0.12 mM second single-stranded oligo DNA (sequence) 5′ ACT CGCAAG CAC CCT ATC A 3′

[0148] Then, 10 μl of a standard DNA (10⁴ copies, 10⁵ copies, 10⁶ copiesand 10⁷ copies/10 μl) or 10 μl of DNA-free TE buffer (10 mM tris-HCl (pH8.0) containing 0.1 mM EDTA) as a negative control was added. Thestandard DNA was a double-stranded DNA consisting of a DNA strand havingthe following sequence and a complementary DNA strand.

[0149] (sequence) 5′ ATT TAG GTG ACA CTA TAG AAT ACA A-(bases 11 to 300in HCV cDNA) 3′

[0150] The reaction solutions were overlaid with 100 μl mineral oil andincubated at 45° C. for 5 minutes. Subsequently, 5 μl of a mixedsolution of the following enzymes was added, and reaction was carriedout at 45° C. for 1 hour.

[0151] 30 U/μl commercially available SP6 RNA polymerase (Takara ShuzoCo., Ltd.)

[0152] 12 U/μl commercially available RNase inhibitor (Takara Shuzo Co.,Ltd.)

[0153] Then, 0.6 μl of a third single-stranded oilgo DNA (2.75 μM) wasadded.

[0154] (sequence) 5′ ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC CAT AGATCA CTC CCC TG 3′

[0155] Subsequently 0.4 μl of AMV reverse transcriptase (37 U/μl, TakaraShuzo Co., Ltd.), as an RNA dependent DNA polymerase and as aDNA-dependent DNA polymerase, was added. After 2 hours of incubation at45° C., 5 μl of the reaction solutions were subjected 2% agarose gelelectrophoresis. After the electrophoresis, the agarose gel was stainedwith 10,000-fold diluted SYBR Green II (Takara Shuzo Co., Ltd.) for 30minutes.

[0156]FIG. 2 shows the pattern on the stained gel. A maximal amount ofthe product was obtained in the presence of magnesium acetate at a finalconcentration of 15 mM.

EXAMPLE 3

[0157] Effects of Potassium Acetate

[0158] The optimum concentration of magnesium acetate for the presentinvention was studied. Reaction solutions having the followingcomposition were prepared, and 14 μl of the reaction solutions werepored into PCR tubes.

[0159] 85.7 mM tris-acetate (pH 8.1)

[0160] 28.9 mM magnesium acetate

[0161] 214.3, 235.7, 257.1 or 278.6 mM potassium acetate

[0162] 21.4% DMSO

[0163] 32.1% sorbitol

[0164] 2.1 mM each of ATP, GTP, CTP and UTP

[0165] 2.1 mM each of dATP, dGTP, dCTP and dTTP

[0166] 214 μg/ml BSA

[0167] 0.12 μM second single-stranded oligo DNA

[0168] (sequence) 5′ ACT CGC AAG CAC CCT ATC A 3′

[0169] Then, 10 μl of a standard DNA (10⁴ copies, 10⁵ copies, 10⁵ copiesand 10⁶ copies/10 μl) or 10 μl of DNA-free TE buffer (10 mM tris-HCl (pH8.0) containing 0.1 mM EDTA) as a negative control was added. Thestandard DNA was a double-stranded DNA consisting of a DNA strand havingthe following sequence and a complementary DNA strand.

[0170] (sequence) 5′ ATT TAG GTG ACA CTA TAG AAT ACA A-(bases 11 to 300in HCV cDNA) 3′

[0171] The reaction solutions were overlaid with 100 μl mineral oil andincubated at 45° C. for 5 minutes. Subsequently, 5 μl of a mixedsolution of the following enzymes was added, and reaction was carriedout at 45° C. for 1 hour.

[0172] 30 U/μl commercially available SP6 RNA polymerase (Takara ShuzoCo., Ltd.)

[0173] 12 U/μl commercially available RNase inhibitor (Takara Shuzo Co.,Ltd.)

[0174] Then, 0.6 μl of a third single-stranded oilgo DNA (2.75 μM) wasadded.

[0175] (sequence) 5′ ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC CAT AGATCA CTC CCC TG 3′

[0176] Subsequently 0.4 μl of AMV reverse transcriptase (37 U/μl, TakaraShuzo Co., Ltd.), as an RNA dependent DNA polymerase and as aDNA-dependent DNA polymerase, was added. After 2 hours of incubation at45° C., 5 μl of the reaction solutions were subjected 2% agarose gelelectrophoresis. After the electrophoresis, the agarose gel was stainedwith 10,000-fold diluted SYBR Green II (Takara Shuzo Co., Ltd.) for 30minutes.

[0177]FIG. 3 shows the pattern on the stained gel. Even when the initialcopy numbers of the target nucleic acid was 10³, the product wasobtained in the presence of potassium acetate at a final concentrationof 120 mM. Thus, a maximal reaction efficiency was obtained in thepresence of potassium acetate at a final concentration of from 110 to130 mM.

EXAMPLE 4

[0178] Effects of Sorbitol

[0179] The optimum sorbitol concentration for the present invention wasstudied. 20 μl of reaction solutions having the following compositionwere pored into PCR tubes.

[0180] 60 mM tris-acetate (pH 8.1)

[0181] 20.3 mM magnesium acetate

[0182] 187.5 mM potassium acetate

[0183] 15% DMSO

[0184] 22.5, 16.8, 13.5 or 11.3% sorbitol

[0185] (final concentration: 15, 11.3, 9 or 7.5%)

[0186] 1.5 mMl each of ATP, GTP, CTP and UTP

[0187] 1.5 mM each of ATP, dGTP, dCTP and dTTP

[0188] 150 μg/ml BSA

[0189] 0.3 μM second single-stranded oligo DNA

[0190] (sequence) 5′ ACT CGC AAG CAC CCT ATC A 3′

[0191] 0.3 μM third single-stranded oligo DNA (sequence) 5′ ATT TAG GTGACA CTA TAG AAT ACA ACA CTC CAC CAT AGA TCA CTC CCC TG 3′

[0192] Then, 5 μl of a standard RNA (10³ copies, 10⁴ copies, 10⁵ copiesor 10⁶ copies/5 μl) or 5 μl of RNA-free TE buffer as a negative controlwas added. The standard RNA had the following sequence.

[0193] (sequence) 5′ GAA UCA AA-(bases 11 to 300 in HCV RNA) 3′

[0194] The reaction solutions were overlaid with 100 μl mineral oil andincubated at 65° C. for 10 minutes, and then the tubes were allowed tostand still in ice-cold water for 5 minutes. Then, 5 μl of a mixedsolution of the following enzymes was added, and reaction was conductedat 45° C. for 4 hours.

[0195] 36 U/μl commercially available SP6 RNA polymerase (Takara ShuzoCo., Ltd.)

[0196] 12 U/μl commercially available RNase inhibitor (Takara Shuzo Co.,Ltd.)

[0197] 9 U/μl AMV reverse transcriptase (Takara Shuzo Co., Ltd.), as anRNA-dependent DNA polymerase and as a DNA-dependent DNA polymerase

[0198] 5 μl of the reaction solutions were subjected to agarose gelelectrophoresis. After the electrophoresis, the gel was stained with10,000-fold diluted SYBR Green II (Takara Shuzo Co., Ltd.) for 30minutes.

[0199]FIG. 3 shows the pattern on the stained gel. The clear bands of300 bp were obtained in the presence of sorbitol at a finalconcentration of 7.5 to 11.3% even when the initial amount of the targetnucleic acid was 10³ copies. Thus, the maximal reaction efficiency wasobtained when the final sorbitol concentration was from 7.5 to 11.3%.

EXAMPLE 5

[0200] Amplification of Double-stranded DNA as Target Nucleic Acid

[0201] The amount of the amplification product from variousconcentrations of a double-stranded DNA as the target nucleic acid wasstudied. A reaction solution having the following composition wasprepared, and 19 μl of the reaction solution was poured into PCR tubes.

[0202] 63.2 mM tris-acetate (pH 8.1)

[0203] 21.3 mM magnesium acetate

[0204] 197.4 mM potassium acetate

[0205] 22.5% DMSO

[0206] 22.5% sorbitol

[0207] 1.6 mM each of ATP, GTP, CTP and UTP

[0208] 1.6 mM each of dATP, dGTP, dCTP and dTTP

[0209] 157.9 μg/ml BSA

[0210] 0.055 μM second single-stranded oligo DNA

[0211] (sequence) 5′ ACT CGC AAG CAC CCT ATC A 3′

[0212] Then, 5 μl of a standard DNA (10³ copies, 10⁴ copies, 10⁵ copiesor 10⁶ copies/5 μl) or 5 μl of RNA-free TE buffer as a negative controlwas added. The standard DNA was a double-stranded DNA consisting of aDNA strand having the following sequence and a complementary DNA strand.

[0213] (sequence) 5′ ATT TAG GTG ACA CTA TAG AAT ACA A-(bases 11 to 300in HCV cDNA) 3′

[0214] The reaction solutions were overlaid with 100 μl mineral oil andincubated at 45° C. for 5 minutes. Subsequently, 5 μl of a mixedsolution of the following enzymes was added, and reaction was conductedat 45° C. for 1 hour.

[0215] 30 U/μl commercially available SP6 RNA polymerase (Takara ShuzoCo., Ltd.)

[0216] 12 U/μl commercially available RNase inhibitor (Takara Shuzo Co.,Ltd.)

[0217] Then, 2.75 μl of a third single-stranded oligo DNA (2.75 μM) wasadded.

[0218] (sequence) 5′ ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC CAT AGATCA CTC CCC TG 3′

[0219] Subsequently, 0.4 μl of AMV reverse transcriptase (37 U/μl,Takara Shuzo Co., Ltd.), as an RNA-dependent DNA polymerase and as aDNA-dependent DNA polymerase, was added. After 2 hours of incubation at45° C., 5 μl of the reaction solutions were subjected to 2% agarose gelelectrophoresis. After the electrophoresis, the agarose gel was stainedwith 10,000 fold diluted SYBR Green II (Takara Shuzo Co., Ltd.) for 30minutes.

[0220]FIG. 5 shows the pattern on the stained gel. Whenever the initialamount of the target nucleic acid was 10³ copies/5 μl or more, bands ofabout 300 bp were detected, and the band density was dependent on theinitial amount of the nucleic acid, which indicates the possibility ofhigh sensitivity assay of a double-stranded DNA as the target nucleicacid.

EXAMPLE 6

[0221] Specific Cutting of RNA Using First Single-stranded OligonucleicAcid and RNaseH

[0222] RNA was cleaved at the 5′ end of a specific nucleic acidssequence by using a first single-stranded oligonucleic acid and RNaseH.

[0223] A reaction solution having the following composition wasprepared, and 7.2 μl of the reaction solution was pored into PCR tubes.

[0224] 40 mM tris-HCl buffer (pH 8.0)

[0225] 4 mM magnesium chloride

[0226] 1 mM dithiothreitol

[0227] 1 μM first single-stranded oligo DNA (11 mer)

[0228] (sequence) 5′ GTC GGG GGG AA 3′

[0229] Then, 1.8 μl of RNA (133 mer, 5.7 μM) was added, 10 minutes ofheating at 65° C. was followed by sudden cooling in ice-cold water. The133 mer RNA had the following sequence.

[0230] (sequence) 5′ (vector sequence) GGG AAA GCU UGC AUG CCU GCA GGUCGA CUC UAG AGG AUC CCC GGG UAC CGA GCU CGA AUU CC (sequence from HCV) UUGG GGG CGA CAC UCC ACC AUA GAU CAC UCC CCU GUG AGG AAC UAC UGU CUU CACGCA GAA AGC GUC UAG C 3′

[0231] (The sequence complementary to the 11 mer DNA is underlined.)

[0232] After 5 minutes of incubation at 37° C., 1 μl of RNaseH (0.01,0.001, 0.0001 or 0.00001 U/μl, Takara Shuzo Co., Ltd.) was added, andreaction was conducted at 37° C. for 1 hour. To the reaction solutions,2 μl gel loading buffer (0.1 M tris-HCl (pH 8.0) containing 60 mM EDTA,0.25% Bromophenol Blue and 40% sucrose) and then 12 μl formamide wereadded. After 5 minutes of incubation at 65° C., 12 μl of the reactionsolutions were subjected to electrophoresis on a polyacrylamide gelcontaining 12% urea.

[0233] The acrylamide gel was washed with water three times for 5minutes each time and stained with 10,000-fold diluted SYBR Green II(Takara Shuzo Co., Ltd.) for 30 minutes.

[0234] Separately, a reaction solution having the following compositionwas prepared, and 10.8 μl of the reaction solution was pored into PCRtubes.

[0235] 40 mM tris-acetate buffer (pH 8.1)

[0236] 37 mM magnesium acetate

[0237] 347 mM potassium acetate

[0238] 22% sorbitol

[0239] 1 mM dithiothreitol

[0240] 0.9 μM first single-stranded oligonucleic acid (11 mer)

[0241] (sequence) 5′ GTC CGG GGG AA 3′

[0242] Then, 1.8 μl of RNA (133 mer, 5.7 μM) was added, and 10 minutesof heating at 65° C. was followed by sudden cooling in ice-cold water.

[0243] After 5 minutes of incubation at 37° C., 1 μl of RNaseH (0.01,0.001, 0.0001 or 0.00001 U/μl, Takara Shuzo Co., Ltd.) was added, andreaction was conducted at 37° C. for 1 hour. To the reaction solutions,2 μl gel loading buffer and then 10 μl formamide were added. After 5minutes of incubation at 65° C., 12 μl of the reaction solutions weresubjected to electrophoresis on a polyacrylamide gel containing 12%urea.

[0244] The acrylamide gel was washed with water three times for 5minutes each time and stained with 10,000-fold diluted SYBR Green II(Takara Shuzo Co., Ltd.) for 30 minutes.

[0245]FIG. 6 shows the results obtained in the respective reactionsolutions having different compositions. When the tris-HCl buffer wasused in the presence of RNaseH at final concentrations of 0.000001 and0.00001 U/μl, the band of the 133 mer disappeared, and bands of 60 to 70mers were detected. The still higher final concentration of RNaseHresulted in further degradation of the 133 mer. On the other hand, whenthe tris-acetate buffer was used in the presence of RNaseH at a finalconcentration of 0.0007 U/μl, the band of the 133 mer disappeared, and aband of a 60 to 70 mer was detected. This band agrees with the RNAfragment obtained when the 133 mer RNA is cut where the firstsingle-stranded oligonucleic acid binds to the 133 mer in terms ofmobility.

[0246] Thus it was possible to cut the target RNA at the 5′ end of thespecific nucleic acids sequence by using the first single-strandedoligonucleic acid and RNaseH.

EXAMPLE 7

[0247] Amplification of RNA as Target Nucleic Acid

[0248] The present invention was performed by using variousconcentrations of a target RNA, and the amplification products wereexamined. A reaction solution having the following composition wasprepared, and 20 μl of the reaction solution was pored into PCR tubes.

[0249] 60 mM tris-acetate (pH 8.1)

[0250] 20.3 mM magnesium acetate

[0251] 187.5 mM potassium acetate

[0252] 22.5% DMSO

[0253] 22.5% sorbitol

[0254] 1.5 mM each of ATP, GTP, CTP and UTP

[0255] 1.5 mM each of dATP, dGTP, dCTP and dTTP

[0256] 150 μg/ml BSA

[0257] 0.3 μM third single-stranded oligo DNA

[0258] (sequence) 5′ ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC CAT AGATCA CTC CCC TG 3′

[0259] 0.3 μM second single-stranded oligo DNA

[0260] (sequence) 5′ ACT CGC AAG CAC CCT ATC A 3′

[0261] Then, 5 μl of a standard RNA (10² copies, 10³ copies, 10⁴ copies,10⁵ copies or 10⁶ copies/5 μl) or 5 μl of RNA-free TE buffer as anegative control was added. The standard RNA had the following sequence.

[0262] (sequence) 5′ GAA UAC AA-(bases 11 to 300 in HCV RNA) 3′

[0263] The reaction solutions were overlaid with 100 μl mineral oil andincubated at 65° C. for 10 minutes, and then the tubes were allowed tostand still in ice-cold water for 5 minutes. Then, 5 μl of a mixedsolution of the following enzymes was added, and reaction was conductedat 45° C. for 4 hours.

[0264] 36 U/μl commercially available SP6 RNA polymerase (Takara ShuzoCo., Ltd.)

[0265] 12 U/μl commercially available RNase inhibitor (Takara Shuzo Co.,Ltd.)

[0266] 9 U/μl AMV reverse transcriptase (Takara Shuzo Co., Ltd.), as anRNA-dependent DNA polymerase and as a DNA-dependent DNA polymerase

[0267] 5 μl of the reaction solutions were subjected to agarose gelelectrophoresis. After the electrophoresis, the agarose gel was stainedwith 10,000-fold diluted SYBR Green II (Takara Shuzo Co., Ltd.) for 30minutes.

[0268]FIG. 7 shows the pattern on the stained gel. As shown in FIG. 7,when the initial concentration of the target RNA was 10³ copies/5 μl ormore, bands of about 300 bp were detected, and the band density wasdependent on the initial amount of the RNA. Thus, it was possible todetect the target RNA with high sensitivity by the present invention.The amount of the amplification product increased with the elapse oftime and was dependent on the initial amount of the target RNA.

EXAMPLE 8

[0269] Identification of Amplification Product

[0270] Amplification products was identified after treatment with DNaseor RNase. The procedure in Example 7 was followed until the use of theenzymes in 5 μl of TE buffer as a negative control and in 5 μl of astandard RNA (10⁵ copies or 10⁶ copies/5 μl). The standard RNA was asfollows.

[0271] (sequence) 5′ GAA UAC AA-(bases 11 to 300 in HCV RNA) 3′

[0272] As the standard DNA, a double stranded-DNA consisting of a DNAstrand having the following sequence and a complementary strand wasused.

[0273] (sequence) 5′ ATT TAG GTG ACA CTA TAG AAT ACA A-(bases 11 to 300in HCV cDNA)-3′

[0274] After the procedure, each reaction solution was incubated at 65°C. for 30 minutes. 6 μl of the three kinds of reaction solutions, thestandard RNA (69 ng/μl) and the standard DNA (6 ng/μl) were pored intothree tubes each. To one of them, 0.6 μl of RNaseA (0.5 mg/ml) wasadded, to another one, 0.6 μl of DNaseI (1 mg/ml) was added, and to theremaining one, nothing was added. The three tubes of each set wereincubated at 37° C. for 1 hour, and 5 μl of each reaction solution wassubjected to 2% agarose gel electrophoresis. After the electrophoresis,the agarose gel was stained with 10,000-fold diluted SYBR Green II(Takara Shuzo Co., Ltd.) for 30 minutes. The compositions of thereaction solutions with the standard RNA (69 ng/μl) and the standard DNA(6 ng/μl) were the same as the composition of the other reactionsolutions obtained by the procedure in Example 7 except that the enzymeswere omitted.

[0275]FIG. 8 shows that the RNA product obtained by DNase I treatment ofthe DNA product from 10⁶ copies of the target standard RNA correspondedto 345 ng of the standard RNA in terms of band density. The band of theRNA product obtained by the DNase I treatment agreed with band of thestandard RNA in terms of mobility.

[0276] On the other hand, the band obtained by amplification of 10⁵copies of the target standard RNA followed by RNaseA treatment of theRNA product almost agreed with the standard DNA in terms of mobility.

[0277] Thus, both the RNA product and the DNA product were synthesizedfrom the target RNA simultaneously.

EXAMPLE 9

[0278] Time Courses of RNA Production and DNA Production

[0279] The time courses of DNA production and RNA production werefollowed. A reaction solution having the following composition wasprepared, and 33 μl of the reaction solution was pored into 10 PCR tubes

[0280] 61 mM tris-acetate (pH 8.1)

[0281] 20.5 mM magnesium acetate

[0282] 189.2 mM potassium acetate

[0283] 21.7% DMSO

[0284] 12% sorbitol

[0285] 15 mM dithiothreitol

[0286] 1.5 mM each of ATP, GTP, CTP and UTP

[0287] 1.5 mM each of dATP, dGTP, dCTP and dTTP

[0288] 151 μg/ml BSA

[0289] 0.3 μM third single-stranded oligo DNA

[0290] (sequence) 5′ ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC CAT AGATCA CTC CCC TG 3′

[0291] 0.3 μM second single-stranded oligo DNA

[0292] (sequence) 5′ ACT CGC AAG CAC CCT ATC A 3′

[0293] Then, 8.3 μl of a standard RNA (10⁶ copies/5 μl) or TE buffer asthe negative control was pored into five tubes. The standard RNA had thefollowing sequence.

[0294] (sequence) 5′ GAA UAC AA-(bases from 11 to 300 in HCV RNA) 3′

[0295] The reaction solutions were overlaid with 100 μl mineral oil andincubated at 65° C. for 10 minutes, and then the tubes were allowed tostand still in ice-cold water for 5 minutes. After 10 minutes ofincubation at 44° C., 8.6 μl of a mixed solution of the followingenzymes was added. After 0, 1, 2, 3 and 4 hours of reaction at 44° C.,one tube for each of the standard RNA and the negative control waswithdrawn and transferred into ice.

[0296] 32.8 U/μl commercially available SP6 RNA polymerase (Takara ShuzoCo., Ltd.)

[0297] 11.8 U/μl commercially available RNase inhibitor (Takara ShuzoCo., Ltd.)

[0298] 8.2 U/μl AMV reverse transcriptase (Takara Shuzo Co., Ltd.), asan RNA-dependent DNA polymerase and as a DNA-dependent DNA polymerase

[0299] 5 μl of the reaction solution in each tube was subjected to 2%agarose electrophoresis. After the electrophoresis, the agarose gel wasstained with 10,000fold diluted SYBR Green II (Takara Shuzo Co., Ltd.)for 30 minutes and photographed. Then, the band densities (OD) in thephotograph were measured with a densitometer.

[0300] The rest of each reaction solution was heated at 65° C. for 20minutes, and 10 μl of each reaction solution were pored into threetubes. To one of them, 1 μl of RNaseA (0.5 mg/ml) was added, to anotherone, 1 μl of DNaseI (1 mg/ml) was added, and to the remaining one, 1 μlof TE buffer was added. All the three tubes of each set were incubatedat 37° C. for 1 hour, and then 5 μl of each reaction solution wassubjected to 2% agarose gel electrophoresis. After the electrophoresis,the gel was stained with 10,000-fold diluted SYBR Green II (Takara ShuzoCo., Ltd.) for 30 minutes and photographed. Then the band densities (OD)in the photograph were measured with a densitometer.

[0301]FIG. 9 shows the time chart of DNA production and RNA productiondetermined from the band densities in the electrophoretogram. The RNAproduction was about 40 times greater than the DNA production. Further,both the RNA production and the DNA production suddenly increased 2hours after the initiation of the reaction. Thus, both RNA productionand DNA production by the method of the present invention showed similaramplification curves.

EXAMPLE 10

[0302] Assay of Amplification Product Using Fourth Single-stranded OligoDNA

[0303] An RNA product in reaction solutions was assayed by measuring thefluorescent signals by using a fourth single-stranded oligo RNA.Firstly, 20 μl of a reaction solution having the following compositionwas pored into 30 PCR tubes.

[0304] 60 mM tris-acetate (pH 8.1)

[0305] 20.3 mM magnesium acetate

[0306] 187.5 mM potassium acetate

[0307] 22.5% DMSO

[0308] 12% sorbitol

[0309] 1.5 mM each of ATP, GTP, CTP and UTP

[0310] 1.5 mM each of dATP, dGTP, dCTP and dTTP

[0311] 150 μg/ml BSA

[0312] 0.3 μM third single-stranded oligo DNA

[0313] (sequence) 5′ ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC CAT AGATCA CTC CCC TG 3′

[0314] 0.3 μM second single-stranded oligo DNA

[0315] (sequence) 5′ ACT CGC AAG CAC CCT ATC A 3′

[0316] Then, 5 μl of a standard RNA (10⁶ copies/5 μl) and TE buffer as anegative control were added to 15 tubes, respectively. The standard RNAhad the following sequence.

[0317] (sequence) 5′ GAA UAC AA-(bases 11 to 300 in HCV RNA) 3′

[0318] The reaction solutions were overlaid with 100 μl mineral oil andincubated at 65° C. for 10 minutes, and then the tubes were allowed tostand still in ice-cold water for 5 minutes. After 10 minutes ofincubation at 44° C., 5 μl of a mixed solution of the following enzymeswas added. After 0, 1, 2, 3 and 4 hours of reaction at 44° C., threetube for each of the standard RNA and the negative control werewithdrawn and transferred into ice.

[0319] 36 U/μl commercially available SP6 RNA polymerase (Takara ShuzoCo., Ltd.)

[0320] 12 U/μl commercially available RNase inhibitor (Takara Shuzo Co.,Ltd.)

[0321] 9 U/μl AMV reverse transcriptase (Takara Shuzo Co., Ltd.), as anRNA-dependent DNA polymerase and as a DNA-dependent DNA polymerase

[0322] 5 μl of the reaction solution in each tube was subjected to 2%agarose electrophoresis. After the electrophoresis, the reactionsolutions in the three tubes withdrawn at each reaction time were mixed,and 5 μl of each mixture was subjected to 2% agarose gelelectrophoresis. After the electrophoresis, the agarose gel was stainedwith 10,000-fold diluted SYBR Green II (Takara Shuzo Co., Ltd.) for 30minutes and photographed.

[0323] To 50 μl of each mixture, 100 μl of measuring buffer having thefollowing composition was added.

[0324] 40 mM tris-acetate (pH 8.1)

[0325] 13 mM magnesium acetate

[0326] 125 mM potassium acetate

[0327] 10 mM dithiothreitol

[0328] 2 U/μl RNase inhibitor

[0329] 37.5 nM fourth single-stranded oligo DNA (hereinafter referred toas YO-271)

[0330] (sequence) 5° CTC GC*G GGG GCT G 3′

[0331] (* indicates the site labeled with the fluorescent intercalativedye, oxazole yellow. The sequence of bases 1 to 11 in the DNA moiety iscomplementary to the sequence of bases 223 to 233 in HCV cDNA. Thefluorescent dye was linked to the DNA moiety as described in NucleicAcids Research, 24(24), 4992-4997(1996) for YO-YPF-271. For thestructure of YO-271, FIG. 19 should be referred to).

[0332] The reaction solutions were incubated at 65° C. for 15 minutesand suddenly cooled in ice-cold water for 5 minutes. Then the reactionsolutions were incubated at 37° C. for 10 minutes and poured intofluorometric cuvettes preheated to 37° C., and the fluorescentintensities were measured at an excitation wavelength of 490 nm and anemission wavelength of 510 nm.

[0333]FIG. 10 shows the results of the electrophoresis, and FIG. 11shows the results of the measurements of the fluorescent signals fromthe fourth single-stranded oligo DNA (YO-271). As is evident from FIG.11, the fluorescent signal from YO-271 increased suddenly in 2 hours forthe standard RNA, whereas there was no increase in the fluorescenceintensity with a negative control. Thus, use of the fourthsingle-stranded oligo DNA enabled to specific assay of the amplificationproduct.

EXAMPLE 11

[0334] Modification of the 3′ End of Fourth Single-stranded Oligo DNA

[0335] During synthesis of a single-stranded RNA in the presence of afourth single-stranded oligo DNA, elongation of the DNA from the 3′ endby the action of a coexisting RNA-dependent RNA polymerase can resultsin increase in a non-specific fluorescent signal. Therefore, the 3′ endof the oligo DNA was modified by treatment with terminal transferase(TdT) (addition of ddTTP), and its effect was examined.

[0336] TdT treatment was conducted in a reaction solution (total volume50 μl) having the following composition at 37° C. for 1 hour.

[0337] 100 mM sodium cacodylate buffer (pH 7.2)

[0338] 1 mM cobalt chloride

[0339] 0.1 mM dithiothreitol

[0340] 0.5 mM ddTTP

[0341] 0.6 U/μl TdT (Takara Shuzo Co., Ltd.)

[0342] 37.3 μM fourth single-stranded oligo DNA (YO-271)

[0343] The modification product was recovered by phenol-chloroformextraction followed by purification from the aqueous layer using acommercial column (chroma spin-10, tradename, Toyobo Co., Ltd.) anddetermined by OD260 measurement.

[0344] The resulting TdT-treated YO-271 and non-treated YO-271 were usedfor measurement of fluorescent signals in the present invention. 50 μlof reaction solutions having the following composition (containingnon-treated YO-271 or TdT-treated YO-271) were pored into 10 tubes,respectively.

[0345] 69 mM tris-acetate (pH 8.1)

[0346] 20.3 mM magnesium acetate

[0347] 187.5 mM potassium acetate

[0348] 21.5% DMSO

[0349] 12% sorbitol

[0350] 15 mM dithiothreitol

[0351] 1.5 mM each of ATP, GTP, CTP and UTP

[0352] 1.5 mM each of dATP, dGTP, dCTP and dTTP

[0353] 150 μg/ml BSA

[0354] 0.3 μM third single-stranded oligo DNA

[0355] (sequence) 5′ ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC CAT AGATCA CTC CCC TG 3′

[0356] 0.3 μM second single-stranded oligo DNA

[0357] (sequence) 5′ ACT CGC AAG CAC CCT ATC A 3′

[0358] 112.5 nM TdT-treated YO-271 or non-treated YO-271

[0359] Then, 12.5 μl of a standard RNA (10⁶ copies/5 μl) or TE buffer asa negative control was added to five tubes, respectively. The standardRNA had the following sequence.

[0360] (sequence) 5′ GAA UAC AA-(bases 11 to 300 in HCV RNA) 3′

[0361] The reaction solutions were overlaid with 100 μl mineral oil andincubated at 65° C. for 10 minutes, and then the tubes were allowed tostand still in ice-cold water for 5 minutes. After 10 minutes ofincubation at 44° C., 12.5 μl of a mixed solution of the followingenzymes was added. After reaction at 40° C. for 0, 1, 2, 3 and 4 hours,tubes were withdrawn and transferred into ice-cold water for thestandard RNA and negative control.

[0362] 34.2 U/μl commercially available SP6 RNA polymerase (Takara ShuzoCo., Ltd.)

[0363] 12 U/μl commercially available RNase inhibitor (Takara Shuzo Co.,Ltd.)

[0364] 8.4 U/μl AMV reverse transcriptase (Takara Shuzo Co., Ltd.) as anRNA-dependent DNA polymerase and as a DNA-dependent DNA polymerase

[0365] A 50 μl aliquot of each reaction solution was mixed with 100 μlof dilution buffer having the following composition, incubated at 44° C.for 5 minutes and transferred into a fluorometric cuvette preheated at44° C., and the fluorescence intensity was measured at an excitationwavelength of 490 nm and an emission wavelength of 510 nm.

[0366] 40 mM tris-acetate (pH 8.1)

[0367] 13 mM magnesium acetate

[0368] 125 mM potassium acetate

[0369] 10 mM dithiothreitol

[0370] 2 U/μl RNase inhibitor

[0371]FIG. 12 and FIG. 13 show the results with the non-treated andTdT-treated YO-271, respectively.

[0372] In the case of the non-treated YO-271, there was an increase inthe fluorescent signal in the presence of the negative control, and thefluorescent signal in the presence of the negative control was notsignificantly different from that in the presence of the standard RNA.On the other hand, in the case of the ddTTP-treated YO-271, there was asignificant difference between the fluorescent signal in the presence ofthe negative control and in the presence of the standard RNA. Thus, forRNA synthesis in the presence of the fourth single-stranded oligo DNA inthe present invention, it is preferred to modify the 3′ end of the DNAso as to prevent elongation of the DNA from the 3′ end by the action ofthe RNA-dependent DNA polymerase

EXAMPLE 12

[0373] RNA or DNA Synthesis in the Presence of Fourth Single-strandedOligo DNA

[0374] The effects of the ddTTP-treated YO-271 prepared in Example 11 onRNA or DNA synthesis in the presence of a fourth single-stranded oligoDNA were examined. 50 μl of a reaction solution having the followingcomposition was pored into 10 tubes.

[0375] 60 mM tri-acetate (pH 8.1)

[0376] 2.3 mM magnesium acetate

[0377] 187.5 mM potassium acetate

[0378] 21.5% DMSO

[0379] 12% sorbitol

[0380] 15 mM dithiothreitol

[0381] 1.5 mM each of ATP, GTP, CTP and UTP

[0382] 1.5 mM each of dATP, dGTP, dCTP and dTTP

[0383] 150 μg/ml BSA

[0384] 0.3 μM third single-stranded oligo DNA

[0385] (sequence) 5′ ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC CAT AGATCA CTC CCC TG 3′

[0386] 0.3 μM second single-stranded oligo DNA

[0387] (sequence) 5′ ACT CGC AAG CAC CCT ATC A 3′

[0388] 112.5 nM TdT-treated YO-271

[0389] Then, 12.5 μl of a standard RNA (10⁶ copies/5 μl) or TE buffer asa negative control was added to five tubes, respectively. The standardRNA had the following sequence.

[0390] (sequence) 5′ GAA UAC AA-(bases from 11 to 300 in HCV RNA) 3′

[0391] The reaction solutions were overlaid with 100 μl mineral oil andincubated at 65° C. for 10 minutes, and then the tubes were allowed tostand still in ice-cold water for 5 minutes. After 10 minutes ofincubation at 44° C., 12.5 μl of a mixed solution of the followingenzymes was added. After reaction at 40° C. for 0, 1, 2, 3 and 4 hours,tubes were withdrawn and transferred into ice-cold water for thestandard RNA and negative control.

[0392] 34.2 U/μl commercially available SP6 RNA polymerase (Takara ShuzoCo., Ltd.)

[0393] 12 U/μl commercially available RNase inhibitor (Takara Shuzo Co.,Ltd.)

[0394] 8.4 U/μl AMV reverse transcriptase (Takara Shuzo Co., Ltd.) as anRNA-dependent DNA polymerase and as a DNA-dependent DNA polymerase

[0395] 5 μl of the reaction solution in each tube was subjected to 2%agarose gel electrophoresis. After the electrophoresis, the agarose gelwas stained with 10,000fold diluted SYBR Green II (Takara Shuzo Co.,Ltd.) for 30 minutes and photographed. The band densities (OD) in thephotograph was measured with a densitometer.

[0396]FIG. 14 shows the results of the electrophoresis, and FIG. 15shows the results of the OD measurement of the electrophoretogram. Inthe presence of the standard RNA, the amplification product increasedwith time, whereas in the presence of the negative control, there was noincrease of the amplification product. Thus, the presence of the fourthsingle-stranded oligo DNA did not inhibit RNA synthesis in the presentinvention.

EXAMPLE 13

[0397] Analysis of the Time Course of the Amount of AmplificationProduct by Using Fourth Single-stranded Oligo DNA

[0398] RNA production was monitored by using the ddTTP-treated YO-271prepared in Example 11 as the fourth single-stranded oligo DNA over aperiod of time. Firstly, 50 μl of a reaction solution having thefollowing composition was pored into 10 tubes.

[0399] 60 nM tris-acetate (pH 8.1)

[0400] 20.3 mM magnesium acetate

[0401] 187.5 mM potassium acetate

[0402] 21.5% DMSO

[0403] 12% sorbitol

[0404] 15 mM dithiothreitol

[0405] 1.5 mM each of ATP, GTP, CTP and UTP

[0406] 1.5 mM each of dATP, dGTP, dCTP and dTTP

[0407] 150 μg/ml BSA

[0408] 0.3 μM third single-stranded oligo DNA

[0409] (sequence) 5′ ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC CAT AGATCA CTC CCC TG 3′

[0410] 0.3 μM second single-stranded oligo DNA

[0411] (sequence) 5′ ACT CGC AAG CAC CCT ATC A 3′

[0412] 112.5 nM TdT-treated YO-271

[0413] Then, 12.5 μl of a standard RNA (10⁶ copies/5 μl) or TE buffer asa negative control was added to five tubes, respectively. The standardRNA had the following sequence.

[0414] (sequence) 5′ GAA UAC AA-(bases 11 to 300 in HCV RNA) 3′

[0415] The reaction solutions were overlaid with 100 μl of mineral oiland incubated at 65° C. for 10 minutes, and then the tubes were allowedto stand still in ice-cold water for 5 minutes. After 10 minutes ofincubation at 44° C., 12.5 μl of a mixed solution of the followingenzymes was added. After reaction at 40° C. for 0, 1, 2, 3 and 4 hours,tubes were withdrawn and transferred into ice-cold water for thestandard RNA and negative control.

[0416] 34.2 U/μl commercially available SP6 RNA polymerase (Takara ShuzoCo., Ltd.)

[0417] 12 U/μl commercially available RNase inhibitor (Takara Shuzo Co.,Ltd.)

[0418] 8.4 U/μl AMV reverse transcriptase (Takara Shuzo Co., Ltd.) as anRNA-dependent DNA polymerase and as a DNA-dependent DNA polymerase

[0419] A 50 μl aliquot of each reaction solution was mixed with 100 μlof dilution buffer having the following composition, incubated at 44° C.for 5 minutes and transferred into a fluorometric cuvette preheated at44° C., and the fluorescence intensity was measured at an excitationwavelength of 490 nm and an emission wavelength of 510 nm.

[0420] 40 mM tris-acetate (pH 8.1)

[0421] 13 mM magnesium acetate

[0422] 125 mM potassium acetate

[0423] 10 mM dithiothreitol

[0424] 2 U/μl RNase inhibitor

[0425]FIG. 16 shows the results of the fluorescence measurement. In thepresence of the standard RNA, the fluorescence intensity increased withthe elapse of time, whereas in the presence of the negative control, thefluorescence intensity did not increase. The fluorescence profile almostagreed with the results of the electrophoretic quantification in Example12. Thus, use of the fourth single-stranded oligo DNA made it possibleto follow the time course of specific amplification (RNA synthesis).

EXAMPLE 14

[0426] Monitoring of a Fluorescent Signal Using Fourth Single-strandedOligo DNA

[0427] The fluorescence intensity of the ddTTP-treated YO-271 preparedin Example 11 in the presence of known concentrations of a target RNAwas monitored. Firstly, 50 μl of a reaction solution having thefollowing composition were pored into 17 PCR tubes.

[0428] 60 mM tris-acetate (pH 8.1)

[0429] 20.3 mM magnesium acetate

[0430] 187.5 mM potassium acetate

[0431] 21.5% DMSO

[0432] 12% sorbitol

[0433] 15 mM dithiothreitol

[0434] 1.5 mM each of ATP, GTP, CTP and UTP

[0435] 1.5 mM each of dATP, dGTP, dCTP and dTTP

[0436] 150 μg/ml BSA

[0437] 0.3 μM third single-stranded oligo DNA

[0438] (sequence) 5′ ATT TAG GTG ACA CTA TAG AAT ACA ACA CTC CAC CAT AGATCA CTC CCC TG 3′

[0439] 0.3 μM second single-stranded oligo DNA

[0440] (sequence) 5′ ACT CGC AAG CAC CCT ATC A 3′

[0441] 112.5 nM TdT-treated YO-271

[0442] Then, 12.5 μl of a standard RNA (10⁴ copies or 10⁵ copies/5 μl)and TE buffer as a negative control were pored into four tubes each.Separately, 12.5 al of the standard RNA (10⁶ copies/5 μl) was pored intofive tubes.

[0443] The standard RNA had the following sequence.

[0444] (sequence) 5′ GAA UAC AA-(bases 11 to 300 in HCV RNA) 3′

[0445] The reaction solutions were overlaid with 100 μl mineral oil andincubated at 65° C. for 10 minutes, and then the tubes were allowed tostand dtill in ice-cold water for 5 minutes. After 10 minutes ofincubation at 44° C., 12.5 μl of a mixed solution of the followingenzymes was added. After reaction at 44° C. for 0, 1 (only for 10⁶copies/5 μl of the standard RNA), 2, 3 and 4 hours, one tube waswithdrawn and transferred into ice-cold water for the standard RNA andthe negative control, respectively.

[0446] 34.2 U/μl commercially available SP6 RNA polymerase (Takara ShuzoCo., Ltd.)

[0447] 12 U/μl commercial available RNase inhibitor (Takara Shuzo Co.,Ltd.)

[0448] 8.4 U/μl AMV reverse transcriptase (Takara Shuzo Co., Ltd.) as anRNA-dependent DNA polymerase and as a DNA-dependent DNA polymerase

[0449] A 50 μl aliquot of each reaction solution was mixed with 100 μlof dilution buffer having the following composition, incubated at 44° C.for 5 minutes and transferred into a fluorometric cuvette preheated at44° C., and the fluorescence intensity was measured at an excitationwavelength of 490 nm and an emission wavelength of 510 nm.

[0450] 40 mM tris-acetate (pH 8.1)

[0451] 13 mM magnesium acetate

[0452] 125 mM potassium acetate

[0453] 10 mM dithiothreitol

[0454] 1 U/μl RNase inhibitor

[0455]FIG. 17 shows fluorescence intensities measured at the respectivereaction times after subtraction of the background fluorescenceintensity. The plot of the fluorescence intensities at the reaction timeof 3 hours against the initial concentrations of the target RNA based onthe fluorescence profiles (FIG. 18) shows concentration-dependentfluorescence enhancement. Thus, a calibration curve which shows thecorrelation between the initial concentration of the target RNA andfluorescence enhancement was obtained on the base of the amplificationcurve obtained by monitoring the fluorescence intensity. Thus, bymeasuring fluorescence intensity for a sample containing the target RNAat an unknown concentration, the initial amount of the target RNA couldbe determined.

[0456] As described so far, the present invention enables assay of asingle-stranded RNA containing a specific nucleic acids sequence in asample at almost constant temperature without repetitious rapid heatingand cooling of reaction solutions. Further, the method of the presentinvention enables high sensitive specific homogeneous assay of a targetnucleic acid without using a support.

[0457] In the present invention, from a trace amount of asingle-stranded RNA in a sample, a double-stranded DNA having a promoterregion for a DNA-dependent RNA polymerase at one end is synthesized, andthe double-stranded DNA gives rise to multiple RNA single strands. Thesynthesized single-stranded RNA participates in the next round ofsynthesis of the double-stranded DNA. Consequently, the single-strandedRNA synthesized in the series of reactions as an intermediatedrastically increases with the elapse of time. However, because the rateof the RNA synthesis and the final amount of the synthesized RNA dependon the amount of the target RNA in the sample, assay of the target RNAis possible by measurement of the amount of the synthesized RNA.

[0458] The fourth single-stranded oligo DNA in the present inventionenhances the fluorescence intensity on hybridization with thesingle-stranded RNA in the reaction solution, and thereby enablesdetermination of the initial amount of the target RNA in the sample bymeasuring the fluorescent intensity of the reaction solution.

[0459] Thus, the assay method of the present invention can be conductedat constant temperature because the reactions proceed through binding ofprimers to the single-stranded RNA and DNA synthesized as intermediates,and can be automated easily because it does not require repetitiousrapid heating and cooling of reaction solutions for priming unlike PCR.Further, in the present invention, the use of the fourth single-strandedoligo DNA makes is possible to monitor RNA synthesis by measuring thefluorescent intensity during the single-stranded RNA. Therefore, it ispossible to provide a simple one-step method for accurate speedyhomogeneous qualitative and quantitative assay of a target RNA usefulfor clinical diagnosis without need of electrophoresis or sandwich assayof the reaction product.

[0460] On the basis of the multiplication of a specific RNA sequence atconstant temperature, the present invention also provides a simplemethod for producing RNA which can be conducted under milder conditionsthan conventional chemical synthesis and RT-PCR.

What is claimed is:
 1. A simple and accurate method for assay of asingle-stranded RNA containing a specific nucleic acids sequence in asample at almost constant temperature by using at least the followingreagents (A) to (I), which comprises a step of adding the reagents (A)to (I) one by one (in any order), in combinations of at least two or allat once and a step of measuring a fluorescent signal in the presence ofthe reagent (I) at least once after addition of at least the reagents(A) to (H); (A) a first single-stranded oligonucleic acid complementaryto a sequence neighboring the 5′ end of the specific nucleic acidssequence in the single-stranded RNA, (B) a second single-stranded oligoDNA complementary to a 3′-end sequence within the specific nucleic acidssequence, (C) an RNA-dependent DNA polymerase, (D) deoxyribonucleosidetriphosphates, (E) a third single-stranded oligo DNA having (1) apromoter sequence for a DNA-dependent RNA polymerase, (2) an enhancersequence for the promoter and (3) a 5′-end sequence within the specificnucleic acids sequence, in this order from the 5′ end, (F) aDNA-dependent DNA polymerase, (G) a DNA-dependent RNA polymerase, (H)ribonucleoside triphosphates, and (I) a fourth single-stranded oligo DNAcomplementary to the specific nucleic acids sequence which is labeled sothat it gives off a measurable fluorescent signal on hybridization witha nucleic acid containing the specific nucleic acids sequence.
 2. Themethod according to claim 1 , wherein the temperature is selected fromthe range of from 35 to 60° C.
 3. The method according to claim 1 ,wherein the first oligonucleic acid as the reagent (A) is a DNA, and themethod further comprises a step of adding an RNaseH and a subsequentstep of deactivating the RNaseH by heating or by addition of aninhibitor prior to addition of the reagent (B).
 4. The method accordingto claim 3 , wherein addition of the reagent (A) is followed bysimultaneous addition of the reagents (B) to (H), and further byaddition of the reagent (I).
 5. The method according to claim 3 ,wherein addition of the reagent (A) is followed by simultaneous additionof the reagents (B) to (I).
 6. The method according to claim 1 , whereinthe first oligonucleic acid as the reagent (A) is a ribozyme or aDNAzyme.
 7. The method according to claim 1 , which further usesdimethyl sulfoxide and/or an enzyme which degrades RNA in a DNA-RNAdouble strand.
 8. The method according to claim 7 , which uses dimethylsulfoxide at a concentration of from 5 to 20%.
 9. The method accordingto claim 7 , wherein the enzyme which degrades RNA in a DNA-RNA doublestrand is the RNA-dependent DNA polymerase as the reagent (C).
 10. Themethod according to claim 1 , wherein an enzyme having both anRNA-dependent DNA polymerase activity and a DNA-dependent DNA polymeraseactivity is used as the reagents (C) and (F) to virtually omit additionof the reagent (C) or the reagent (F).
 11. The method according to claim10 , wherein the enzyme is avian myoblastome virus polymerase.
 12. Themethod according to claim 1 , wherein the second and third oligo DNAs asthe reagents (B) and (E) are used at concentrations of from 0.02 to 1μM.
 13. The method according to claim 1 , wherein the DNA-dependent RNApolymerase as the reagent (G) is at least one enzyme selected from thegroup consisting of phage SP6 polymerase, phage T3 polymerase and phaseT7 polymerase.
 14. The method according to claim 1 , wherein the fourtholigo DNA as the reagent (I) is a DNA which is linked to a fluorescentintercalative dye so that the fluorescent intercalative dye changes itsfluorescence characteristic on hybridization of the DNA with anothernucleic acid by intercalating into the resulting double strand.
 15. Themethod according to claim 1 or 14 , wherein the fourth oligo DNA as thereagent (I) is a DNA which has a 3′-end sequence uncomplementary to thespecific nucleic acids sequence or has a modified 3′ end.
 16. The methodaccording to claim 1 , which further comprises a step of detecting orquantifying the single-stranded RNA in the sample based on the measuredfluorescent signal or change in the measured fluorescent signal.
 17. Themethod according to claim 1 , wherein all the reagents arechloride-free.
 18. The method according to claim 1 , which further usesan acetate.
 19. The method according to claim 18 , wherein the acetateis magnesium acetate at a concentration of from 5 to 20 mM or potassiumacetate at a concentration of from 50 to 200 mM.
 20. The methodaccording to claim 1 , which further uses sorbitol.
 21. A simple methodfor producing a nucleic acid having a specific nucleic acids sequence atalmost constant temperature by using at least the following reagents (A)to (H), which comprises a step of adding the reagents (A) to (G) one byone (in any order), in combinations of at least two or all at once to asingle-stranded DNA having (1) a promoter sequence for a DNA-dependentRNA polymerase, (2) an enhancer sequence for the promoter and (3) thespecific nucleic acids sequence, in this order from the 5′ end or to adouble-stranded DNA consisting of the single-stranded DNA and acomplementary DNA strand and a step of measuring a fluorescent signalfrom the reagent (H) at least once after addition of at least thereagents (A) to (G); (A) a single-stranded oligo DNA complementary to a3′-end sequence within the specific nucleic acids sequence, (B) anRNA-dependent DNA polymerase, (C) a DNA-dependent DNA polymerase, (D)deoxyribonucleoside triphosphates, (E) a DNA-dependent RNA polymerase,(F) ribonucleoside triphosphates, (G) a single-stranded DNA having (1) apromoter sequence for a DNA-dependent RNA polymerase, (2) an enhancersequence for the promoter and (3) a 5′-end sequence within the specificnucleic acids sequence, in this order from the 5′ end, (H) a fourthsingle-stranded labeled oligo DNA complementary to the specific nucleicacids sequence which gives a measurable fluorescent signal onhybridization with a nucleic acid containing the specific nucleic acidssequence.
 22. The method for producing a single-stranded RNA having aspecific nucleic acids sequence according to claim 21 , wherein a DNaseis added when the measured fluorescent signal or change in the measuredfluorescent signal indicates production of a predetermined amount of thespecific nucleic acids sequence.
 23. The method for producing adouble-stranded DNA consisting of a DNA strand having a specific nucleicacids sequence and a complementary DNA strand according to claim 21 ,wherein an RNase is added when the measured fluorescent signal or changein the measured fluorescent signal indicates production of apredetermined amount of the specific nucleic acids sequence.
 24. Areagent set for performing the method according to claim 1 or 21 , whichcomprises at least a first reagent containing the first single-strandedoligonucleic acid, a second reagent containing tris-acetate, magnesiumacetate, potassium acetate, sorbitol and dimethyl sulfoxide, a thirdreagent containing dithiothreitol, deoxyribonucleoside triphosphates,ribonucleoside triphosphates, bovine serum albumin, the secondsingle-stranded oligo DNA and the third single-stranded oligo DNA, afourth reagent containing an RNA-dependent DNA polymerase, aDNA-dependent DNA polymerase, a DNA-dependent RNA polymerase and anRNase inhibitor and a fifth reagent containing the fourthsingle-stranded oligo DNA.
 25. A reagent set for performing the methodaccording to claim 1 or 21 , which comprises at least a first reagentcontaining the first single-stranded oligonucleic acid, a second reagentcontaining tris-acetate, magnesium acetate, potassium acetate, sorbitoland dimethyl sulfoxide, a third reagent containing dithiothreitol,deoxyribonucleoside triphosphates, ribonucleoside triphosphates, bovineserum albumin, the second single-stranded oligo DNA, the thirdsingle-stranded oligo DNA and the fourth single-stranded oligo DNA and afourth reagent containing an RNA-dependent DNA polymerase, aDNA-dependent DNA polymerase, a DNA-dependent RNA polymerase and anRNase inhibitor.
 26. A reagent set for performing the method accordingto claim 1 or 21 , which comprises at least a first reagent containingthe first single-stranded oligonucleic acid, a second reagent containingtris-acetate, magnesium acetate, potassium acetate, sorbitol anddimethyl sulfoxide, a third reagent containing dithiothreitol,deoxyribonucleoside triphosphates, ribonucleoside triphosphates, bovineserum albumin, the second single-stranded oligo DNA and the thirdsingle-stranded oligo DNA, a fourth reagent containing the fourthsingle-stranded oligo DNA, an RNA-dependent DNA polymerase, aDNA-dependent DNA polymerase, a DNA-dependent RNA polymerase and anRNase inhibitor.
 27. A reagent for performing the method according toclaim 1 or 21 , which comprises at least the first single-strandedoligonucleic acid, the second single-stranded oligo DNA, the thirdsingle-stranded oligo DNA, the fourth single-stranded oligo DNA, anRNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, aDNA-dependent RNA polymerase, deoxyribonucleoside triphosphates,ribonucleoside triphosphates, tris-acetate, magnesium acetate, potassiumacetate, sorbitol, dimethyl sulfoxide, dithiothreitol, bovine serumalbumin and an RNase inhibitor.
 28. The reagent set or reagent accordingto any one of claims 24 to 27 , wherein an enzyme having both anRNA-dependent DNA polymerase activity and a DNA-dependent DNA polymeraseactivity is used at least as the RNA-dependent DNA polymerase and as theDNA-dependent DNA polymerase.